Project Summary Huntington?s disease (HD) is a severe neurological disorder that is characterized by the dysfunction and death of specific brain neurons. HD is directly associated with abnormal function of the protein huntingtin (Htt) in individuals with a polyglutamine expansion at the N-terminus (Htt-e). Htt functions as a protein scaffold and one aspect of Htt-e malfunction is alterations in its interactions with its binding partners, including the pre-mRNA processing protein 40 homolog A (Prp40A). Interestingly, previous studies have shown that the activities of both Htt and Prp40A are modulated by intracellular Ca2+ sensors in a Ca2+-dependent manner. One Htt-binding Ca2+ sensor is the ubiquitous calmodulin (CaM), whose interaction modulates the activation of transglutaminase 2 cross-linking of Htt-e that leads to aggregation in HD brain. Centrin, another EF-hand Ca2+ sensor closely related to CaM, has been found to colocalize with Htt at the centrosome and in the photoreceptor cilium, and both proteins have a regulatory role in ciliogenesis. Yeast Prp40 was found to interact with yeast CaM in a protein microarray experiment. Our research group recently reported that a hPrp40A peptide motif interacts with human centrin 2 (Cen2) in a Ca2+-dependent manner. Together, the results summarized here suggest that Htt, hPrp40A, CaM, and Cen2 form a network that is modulated by intracellular Ca2+ signals and may be involved in HD pathogenesis. In order to explore this intriguing possibility and ultimately determine if this network is a viable target for developing HD therapeutics, it will be necessary to better understand the molecular basis and functional outcomes of these interactions. Here, I propose a first step to fill this critical gap in knowledge by carrying out biophysical and structural analysis of the interaction between hPrp40A and the EF-hand Ca2+ sensors hCaM and hCen2. In Aim 1 I will complete the analysis of the interaction of hPrp40A and hCen2 by obtaining a high- resolution structure of the complex by X-ray crystallography. Aim 2 will validate the physical interaction of hPrp40A and hCaM, measure the binding affinity by isothermal titration calorimetry and determine the high- resolution X-ray crystal structure of their complex. In Aim 3, I will generate a structural model of the hCaM- hPrp40A-hCen2 ternary complex using a combination of small angle X-ray scattering, negative-stain electron microscopy, and computational modeling. These results will be valuable for subsequent analyses of how this protein network functions, evaluation of its role in HD, and set the stage for my long-term research goal of determining if the Htt-Prp40A-Cen2-CaM network is a viable target for developing an HD therapy.